Project Summary/Abstract The process of liquid-liquid phase separation (LLPS) drives formation of numerous membrane-less organelles in cells, the largest of which is the nucleolus. The nucleolus, through its multi-layered, dense liquid structure, controls ribosome biogenesis and also serves as a center for cellular stress signaling involving the tumor suppressors p53 and Arf. The nucleolus is a site of toxicity for di-peptide repeat (DPR) polypeptides observed in the cells of amyotrophic lateral sclerosis (ALS) patients. In 2016 & 2018, we reported that the abundant nucleolar protein, Nucleophosmin (NPM1), undergoes LLPS with ribosomal RNA (rRNA) and ribosomal proteins (r-proteins), and with itself. We view NPM1 as a master organizer of the Granular Component (GC), the outer region of the nucleolus, wherein rRNA assembles with r-proteins to form ribosomal subunits. Pre- rRNA is transcribed in the center of the nucleolus and then captured through LLPS with the protein, Fibrillarin (FIB1), in the Dense Fibrillar Component (DFC). After processing in the DFC, rRNA fluxes outwards and is captured in the GC through LLPS with NPM1. Simultaneously, r-proteins are sequestered in the GC through LLPS with NPM1. Our working model of ribosome assembly is that NPM1 ?escorts? rRNA from the DFC into the GC, where it encounters NPM1-escorted r-proteins moving oppositely. We propose that LLPS with NPM1 and FIB1 concentrates and co-localizes ribosomal components to assemble via a molecular hand-off model, where NPM1 enhances rRNA:r-protein encounters, facilitating their binding, co-folding and ribosome subunit assembly. Further, we propose that the Arf tumor suppressor functions within this dense liquid microenvironment to independently modulate p53 activity and ribosome biogenesis, and that this microenvironment is dramatically altered by the toxic DPRs observed in ALS. We view LLPS-prone proteins through the lens of polymer theory, and apply our expertise with intrinsically disordered proteins to discover the molecular mechanisms that enable nucleolar components (e.g., proteins and RNA) to behave collectively through LLPS to form micron-scale, liquid nucleoli. We seek to understand how the nature of protein and RNA inter-molecular interactions, often involving disordered and multivalent protein regions, influences the material properties of the nucleolus and, consequently, ribosome assembly. The emerging field of biological fluids requires new conceptual frameworks that blend the fields of structural and cell biology with concepts from fluid physics and polymer theory. We will apply a wide-range of structural, biophysical and biochemical, microrheology and cell biology techniques, to relate the molecular properties of proteins and RNA to the material properties of phase separated bodies. Essentially, we seek to establish disorder/multivalency-phase separation-function relationships, using the nucleolus as a model system.